Physical examination or diagnosis of disease states has been conventionally carried out by collecting a large amount, such as several cubic centimeters, of blood from a patient, and using the measurement values obtained from large scale automated blood analysis apparatuses in the analysis of the collected blood. In general, such automated analysis apparatuses are available in medical institutions such as hospitals, and are large in size, and their operation is limited to those who are technically qualified.
However, in recent years, there is a growing tendency for the development and practicalization of a new device which can instantly inform the health status of an examinee, by arranging various analytic apparatuses such as sensors on a substrate having a size of a few millimeters to a few centimeters at maximum at each side of rectangle, as a result of application of the microprocessing technique used in the production of highly advanced semiconductor devices, and introducing a body fluid of the examinee, such as blood. Development of such low-priced devices allows an attempt to reduce the ever-increasing health insurance benefits by enabling daily health management of aged people at home in the on-coming aging society, and so on. Further, such devices are expected to have various social effects, such as that in the field of emergency medicine, if the presence or absence of any infectious disease (hepatitis, acquired immune deficiency, etc.) in an examinee, etc. could be quickly judged by using the device, appropriate action would be possibly taken in response; thus, much attention is being paid to this technical field. As such, instead of the conventional automated analytic apparatuses, small scaled and convenient blood analysis method and blood analysis apparatus, which are aimed at performing blood analysis personally at home, are under development (See, for example, Patent Document 1).
Patent Document 1: JP-A No. 2001-258868
FIG. 1 shows an example of the micromodularized blood analysis apparatus described in Patent Document 1. Symbol 101 represents a lower substrate of the blood analysis apparatus, on which at microgroove flow channel (microcapillary) 102 formed by etching is formed. Over this lower substrate 101, an upper substrate (not shown in the figure) of almost the same size is glued together to seal the groove flow channel 102 from the outside.
Along the flow channel 102, there are sequentially formed a blood collecting means 103, a plasma separating means 104, an analyzing means 105 and a transporting means 106, from the uppermost stream part to the lowermost stream part. The blood collecting means 103 at the forefront part of the flow channel is equipped with a hollow blood collecting needle 103a, and this blood collecting needle 103a is pricked into the body and is used as an inlet for blood into the substrate. The separating means 104 is a bend formed in the middle of the flow channel 102 and consists of, for example, a U-shaped microcapillary. The collected blood is led to this U-shaped microcapillary, and then the substrate is subjected to acceleration in a certain direction by a centrifuge, thereby the blood corpuscle component being precipitated at the lowest part of the U-shape, and the blood plasma being separated as supernatant. The analyzing means 105 are sensors for measuring the pH value and the respective concentrations of oxygen, carbon dioxide, sodium, potassium, calcium, glucose, lactic acid and the like in the blood.
The transporting means 106 which is disposed at the lowermost stream of the flow channel is intended to transport the blood in the microcapillary by electroosmotic flow, and consists of electrodes 107 and 108, and a portion of flow channel 109 connecting the two electrodes. The electroosmotic flow which is generated when voltage is applied between the electrodes, transports the buffer solution that has been preliminarily filled in the flow channel, to the downstream side of the flow channel, and the suction force generated thereby allows uptake of the blood from the collecting means 103 at the forefront part of the flow channel 102 into the substrate. This suction force also drives the blood plasma obtained by centrifuge into the analyzing means 105.
Symbol 110 represents an output means for taking out information from the analyzing means, and consists of electrodes and the like. Symbol 111 represents a control means for controlling the collecting means, plasma separating means, analyzing means, transporting means and output means, as necessary.
The blood collected from the collecting means 103 is separated into the plasma component and the corpuscle component at the separating means 104, and this blood plasma is led to the analyzing means 105, where the pH value and the respective concentrations of oxygen, carbon dioxide, sodium, potassium, calcium, glucose, ureic nitrogen, creatinine, lactic acid and the like in the blood plasma are measured. The transportation of blood between the respective means is carried out by the transporting means 106 having an ability for pumping, such as by means of electrophoresis or electroosmotic phenomenon or the like. In FIG. 1, the downstream region of the flow channel 102 is branched into 5 subregions, and each of these subregions has the analyzing means 105 and the transporting means 106 formed therein.
In many cases, glass materials such as quartz have been used for the substrate of such blood analysis apparatus. However, in consideration of their suitability for mass production of the apparatus at low costs, and the ease of disposal after use, resin materials have been recently put to use.
The conventional blood analysis apparatus illustrated in FIG. 1 requires a transporting means such as an electroosmotic pump 106 for introducing a blood sample into the apparatus. In every substrate, after obtaining blood plasma by centrifuging the introduced blood, it is required to re-operate the electroosmotic pump 106 in order to transport the blood plasma to the analyzing means 105. Furthermore, when the analyzing means are, in particular, sensors established on the basis of the principles of electrochemistry, these sensors need to be calibrated in advance using a calibration solution. That is, before leading the blood plasma into the sensors, these sensors should be immersed in the calibration solution to carry out calibration of the sensors, and the calibration solution should be discharged from the analyzing means after calibration. Such transportation of the calibration solution also requires the transporting means such as a pump.
For the transporting means, it can be considered to use electroosmotic pumps formed on the same substrate as shown in FIG. 1, or negative pressure pumps installed outside the substrate. These transporting means allow transportation of the blood, or the blood plasma, calibration solution and the like, by pneumatic transportation or suction. Here, in order to transport a desired liquid to a desired site in the blood analysis apparatus, it is necessary to precisely control the suction force of the transporting means and the like. In this regard, sensors for liquid position should be newly installed in the blood analysis apparatus or outside the apparatus, but addition of such controlling instruments or position sensors has been causing a problem of making the apparatus expensive.
When the analyzing means are sensors established on the basis of the principles of electrochemistry, the sensors should be calibrated with a calibration solution (reference solution) containing the components to be tested at known concentrations, and then this calibration solution should be discharged from the analyzing means. However, even after discharging the calibration solution, there may be residual calibration solution remaining on the surface of the analyzing means or flow channel means, depending on the wettability of the surface. As described above, since the blood analysis apparatus being presently discussed is intended to analyze the concentrations of various chemicals present in a trace amount, such as about a few microliters, of blood, the size of the means constituting the apparatus, such as the flow channel means, is diminished. In general, when the size of an object is decreased, the ratio S/V of the surface area (S) and volume (V) increases, and this implies that the effect of the surface is significantly exhibited. Therefore, there has been a problem that even though the amount of the calibration solution remaining on the surface of the flow channel means or analyzing means is a trace amount, that amount of the residual calibration solution has an impact on the fluctuation of the concentrations of measured chemicals in an analysis apparatus in which the amount of the blood plasma introduced is minimal. To this end, it is required that only after the calibration solution is certainly discharged out of the analyzing means, the blood plasma is introduced to the analyzing means.
In consideration of the above-described circumstances, the present inventors have suggested a blood analysis apparatus for performing plasma separation in the flow channel by centrifuge operation, which enables conveying of the blood, blood plasma and calibration solution in the apparatus without using pumps or the like, and which enables precise analysis by certainly discharging the calibration solution from the sensor part (see, for example, Patent Document 2).
Patent Document 2: Japanese Patent Application No. 2003-040481
FIG. 2 illustrates an example of the blood analysis apparatus described in Patent Document 2 (unpublished). Symbol 201 represents an upper substrate in which a flow channel is formed, and symbol 202 represents a lower substrate in which sensor electrodes 203 or electrode terminals 204 for taking the sensor signals out of the system are formed. The upper substrate 201 is equipped with a blood collecting needle 205, and the collected blood is transported from an opening for suction and pneumatic transportation 208 to a blood reservoir 207 through a guiding flow channel 206 by means of an external pump (not shown in the figure). A flow channel 209 and flow channel 210 are connected to opening holes 211 and 212, respectively, which are formed on a side wall of the upper substrate 201. However, upon the suction of blood, the opening holes 211 and 212 are closed by a holder (not shown in the figure) on which the blood analysis substrate is mounted. Likewise, a calibration solution reservoir 213 stores a calibration solution introduced from the opening for suction and pneumatic transportation 208.
Exemplary operation of this already-suggested blood analysis apparatus substrate will be described in the following. First, when the blood analysis apparatus substrate is centrifuged around the central axis of the first centrifugal force 214, the calibration solution in the calibration reservoir 213 is taken into a plurality of sensor grooves 217 housing a plurality of sensors 203, through guiding flow channels 215 and 216. After calibration of the sensors 203, the blood analysis apparatus substrate is rotated 90 degrees clockwise and mounted on the centrifuge. That is, when the substrate is centrifuged around the central axis of the second centrifugal force 218 which is located on the left side of FIG. 2, the calibration solution filling the sensor grooves 217 flows through guiding flow channels 216 and 219 and is stored in a calibration solution waste reservoir 220.
Then, the blood analysis apparatus substrate is rotated 90 degrees counterclockwise and mounted on the centrifuge. That is, when the substrate is centrifuged around the central axis of the first centrifugal force, 214, the blood from the blood reservoir 207 is conveyed to the sensor grooves 217 through a guiding flow channel 221. When the centrifugal force is continuously applied as such, the corpuscle component in the blood is fractionated in the direction to which gravity is applied, that is, down to the lower side of the sensor grooves 217, and the plasma component is separated to the upper side of the sensor grooves 217 as a supernatant. A group of sensors 203 are disposed in this region, so that the pH value and the respective concentrations of oxygen, carbon dioxide, sodium, potassium, calcium, glucose, lactic acid and the like in the blood are measured by an external measuring instrument through a plurality of electrode terminals 204 connected to the respective sensors.
This already-suggested blood analysis apparatus can be subjected to centrifuge operation in two different directions, and allows conveyance of the calibration solution in the calibration solution reservoir to the sensor part by centrifuge operation in the first centrifugal direction, and after the calibration of sensors, certain discharge of the calibration solution from the sensor part by centrifuge operation in the second centrifugal direction. After the discharge of the calibration solution, centrifuging in the first centrifugal direction allows conveyance of the blood in the blood reservoir to the sensor part, as well as separation of the blood into the corpuscles and the plasma.
However, even with these advantageous, the blood analysis apparatus was found to have unignorable problems in carrying out blood analysis in short time due to the use of centrifugal force.
It is definitely critical that the measurement time to be taken by a blood analysis apparatus chip should be as short as possible. In the present blood analysis apparatus, the distance from the central axis of centrifugal force to the center of the chip is 5 cm, and the time normally required for infusion or discharge of the calibration solution is about 1 second, even with a small centrifugal force of 3000 rpm or less. However, in order to separate the corpuscles and the plasma in the blood in a few seconds to a few minutes, a centrifugal force of at least 4000 rpm or greater is required at the region of corpuscle separation. FIG. 12 shows the relationship between the speed of rotation (rpm) and the acceleration (G) at this time, in which 3000 rpm corresponds to application of gravitational acceleration of 500 G, and 4000 rpm corresponds to application of gravitational acceleration of 1000 G.
It was found that the output of the sensors is reduced by the centrifuge operation upon the separation of blood corpuscles and blood plasma. For example, when a calibration solution (containing 137 mM sodium ions) was measured with a sodium ion sensor, the output voltage was affected by the speed of rotation (rpm) during centrifuge, as shown in FIG. 3. The sensor output indicated a stable value of about 200 mV up to a speed of rotation of about 3000 rpm; however; at a higher speed of rotation, the sensor output showed a tendency to decrease, and at the same time, the distribution of the value increased. In the present measurement, sensors exhibiting a stable value of about 200 mV up to 1000 rpm were provided and used for the respective rotation tests. Although not particularly mentioned, the same tendency was observed in the measurement of potassium ions.
In a sodium ion concentration measuring sensor, bis(12-crown-4) of the ion sensing membrane capturing sodium ions, and an anion scavenging agent which takes the role of preventing anions in the blood plasma from penetrating into the sensing membrane are mixed with PVC (polyvinyl chloride), and this mixture is immobilized on a carbon electrode to be used as the sensor. Here, in order to make it easier to introduce sodium ions into the sensing membrane, a large amount of plasticizer is mixed into the PVC. When the centrifugal force at 7000 rpm is estimated from the weight of one sensor, the force exerted on the sensor is in the order of pico-newtons. However, it is conjectured that the cause of such reduction in the sensor output at the high speed of rotation might involve deformation of the PVC membrane, which includes the ion sensing membrane and contains the plasticizer, on the carbon electrode due to the strong centrifugal force, thereby a part of the PVC membrane delaminating from the carbon electrode and allowing water penetration. It can be considered to harden the membrane by altering the membrane composition and to strengthen the immobilization of membrane onto the carbon electrode; but, hardening of the membrane may lead to loss of the original characteristics of the electrochemical sensor.